1. Technical Field
This invention relates in general to products and methods for the therapeutic and prophylactic treatment of mammals, to control the phenotypic expression of cancer, and to the production of products and methods for testing for carcinogenicity of environmental substances.
2. Background Art
For many years, cancer in its numerous forms, has been a frightful bane to human society. In many cases, the condition is discovered when the pathological condition has advanced to the point that the patient's life cannot be saved, and the fatal progress of the disease cannot be reversed.
Some cancerous conditions, if diagnosed and treated in a timely fashion, may be arrested with the life of the patient thereby prolonged. It is the hope of this outcome that motivates cancer patients to spend substantial amounts of money on varying forms of cancer treatment. Because cancer attacks the organism at the elementary, cellular level, through the uncontrolled proliferation of cells, the treatment of cancer has been, historically, dramatic and often destructive of the organism.
In effect, many treatments for cancer, because they are intended to function dramatically at the cellular level, sometimes themselves destroy healthy cells. Often, destruction of a sufficient number of healthy cells has contributed to, if not caused, death of the patient. Nevertheless, since the patient has only the prospect of imminent death as an alternative, drastic, expensive and life-threatening treatments, such as X-radiation and chemotherapy, have been employed.
Even when such treatment is successful, the cancer patient remains disabled and ill for significant periods of time after treatment has ceased. Generally, the patient requires hospitalization, not only during the period of treatment, but for significant times thereafter.
For these reasons, the spectre of cancer has caused great fear in human society. The economic impact, in terms of cost of medical care, combined with the disabling effect of the disease on its sufferers have made the search for reliable methods of diagnosing not only the disease but a predisposition to it, and of treating the disease and/or causing its predisposition very significant.
Much of the focus of cancer research has been on the diagnosis and treatment of the condition. In recent years, because of advances in knowledge of biochemical processes at the cellular and subcellular level, attention has been directed to methods, not only for diagnosing and treating cancer, but also for discovering a predisposition for cancer in the organism. In order to determine such a predisposition, studies have been done to determine the mechanisms in the body for suppressing cancer.
In these studies, “cancer suppression” was originally defined by a loss of tumorigenicity observed in fusion cells made with tumor cells and normal fibroblasts, lymphocytes or keratinocytes. The effect was presumed to be mediated by dominant suppressive factors in normal cells, Nature, 223:363 (1969) and J. Cell Sci., 8:659 (1974).
Evidence indicated that these factors were in part genetic since a correlation existed between suppression of tumorigenicity and the presence of certain chromosomes in fused cells, Adv. Viral Oncol., 6:83 (1987). For example, Wilms tumor, a childhood tumor of the kidney, is thought to arise by inactivation of a gene on chromosome 11. Using the technique of microcell fusion-mediated transfer of single chromosomes, it has been demonstrated that introduction of a normal chromosome 11 into Wilms tumor cells suppressed their tumorigenicity. On the other hand, the introduction of chromosomes X and 13 did not have this effect, Science, 236:175 (1987).
However, since entire human chromosomes were transferred, cancer suppression could not be attributed to molecularly defined genetic elements. In addition, the transfet of entire human chromosomes may present significant problems when attempted on a basis other than experimental. The preparation of suitable chromosomes for therapeutic applications is very exacting, time-consuming and expensive. As a result, such a technique has not been found to be acceptable for many applications. For these reasons, it would be highly desirable to have a method of accomplishing cancer treatment biotechnically, both therapeutically and prophylactically, which would overcome such problems associated with the introduction of chromosomes to the patient.
Another meaning for cancer suppressing genes arose in connection with genetic studies on certain childhood neoplasms, Cancer, 35:1022 (1975) and Nature, 316:330 (1985) and adult tumor syndromes Nature, 328:614 (1987); ibid, 332:85 (1988); ibid, 329:246 (1987); ibid, 322:644 (1986). Genes contributing to the formation of these tumors appear to be oncogenic by loss of function, rather than activation, as with the classical oncogenes, Science, 235:305 (1987); ibid, 238:1539 (1987); Nature, 323:582 (1986); Cancer Res., 46:1573 (1986).
Retinoblastoma, a childhood eye cancer, provides the prototypic example, .J. Cell. Biochem., in press (1988). Refined cytogenetic analyses, Am. J. Dis. Child., 132:161 (1978); Science, 208:1042 (1980); J. Med. Genet., 21:92 (1984) and study of restriction fragment length polymorphisms (RFLPs), Nature, 305:779 (1983) suggested that retinoblastoma may result from loss of a gene locus, called RB or RLB-1, located in chromosome band 13q14. A gene from this region possessing properties consistent with the RB gene, has been molecularly cloned, Nature, 323:643 (1986); Science, 235:1394 (1987), and ibid, 236:1657 (1987). Expression of this gene as a 4.7 kb mRNA transcript was found in all normal tissues examined, but was undetectable or altered in retinoblastoma cells, Science, 235:1394 (1987), and the mutations within the RB gene have been identified in many cases Science, 236:1657 (1987), and Proc. Natl. Acad. Sci., 85:2210 (1988) and ibid, 85:6017 (1988). This data suggested that the cloned RB gene was tentatively identified.
A protein product of the RB gene was previously identified as a nuclear phosphoprotein of about 110 kd (pp110RB) using antibodies generated against selected epitomes predicted from the RB cDNA sequence, Nature, 329:642 (1987).
In light of the evidence establishing the cancer suppression properties of the RB gene, work has been done to utilize the RB gene in the determination of the susceptibility to retinoblastoma as a diagnostic tool. These diagnostic methods and products are disclosed in pending U.S. patent application Ser. No. 108,748, which describes cloning, isolation, identification and sequencing of the RB gene. In addition, said patent application also discloses the method of use of the cloned retinoblastoma gene cDNA as a tool for diagnosing retinoblastoma, osteosarcoma and fibrosarcoma.
Additionally, U.S. patent application, Ser. No. 098,612 (now U.S. Pat. No. 4,942,123 Jul. 17, 1990), discloses a phosphoprotein ppRB110 which is primarily located in the cell nucleus and has DNA binding activity. As with RB mRNA, this protein was detected in many types of cultured human cells. pp110RB has been shown to form a tight association with large T antigen and E1A, the transforming proteins of DNA tumor viruses SV40 and adenovirus respectively, Nature, 334:124 (1988); Cell, 54:275 (1988). The RB gene product, or a complex containing it, has been found to have DNA binding activity, Nature, 329:642 (1987). These studies indirectly suggested that pp110RB has a role in regulating the expression of other cellular genes, and may also mediate the oncogenic effects of some viral transforming proteins.
Much of the current cancer research is directed toward the detection and a predisposition in the organism toward development of cancer. Therefore, it would be highly desirable if a prophylactic method of cancer treatment existed so that tumorigenesis could be arrested before its inception or, even more importantly, foreclosed from development altogether.
In this regard, in pending U.S. application, Ser. No. 091,547, there is described methods for using cloned human esterase D cDNA as a genetic marker as a diagnostic tool for retinoblastoma, Wilson's disease, and other hereditary or acquired disceases controlled by genes located at the 13 chromosome 13q:1411 region. The patent application discloses an esterase D cDNA probe for cloning the retinoblastoma gene, the use of the cloned human esterase D cDNA as a prognostic tool for determination of genetic predisposition to retinoblastoma or Wilson's disease.
Thus, although significant advances are being made in the development of prognostic tools for determination of the genetic predisposition to cancer, therapeutic and prophylactic treatment of cancer still present the serious foregoing-mentioned limitations. In this regard, the prognostic tool is extremely useful in screening a population, to determine which persons may have a predisposition toward cancer. Thus, once a person is determined by means of the diagnostic tool as having such a predisposition, the person can be monitored at short intervals for the early signs and symptoms of cancer. If such is found, appropriate procedures, such as surgery, can be undertaken at an early date.
However, while the use of diagnostic tools for the predisposition toward cancer is highly advantageous, the knowledge of such a predisposition is not helpful in the situation where a patient is determined by conventional examination techniques to have, for example, an advanced stage of cancer. In these cases, conventional procedures and treatments have not proven to be entirely satisfactory.
Therefore, it would be highly desirable to have prophylactic and/or therapeutic treatments for cancer, by utilizing biotechnical techniques. Moreover, it would be important to have such biotechnical modalities, which are effective for many different forms of cancer, with little or no side effects. It would also be desirable to have techniques for proving that certain environmental substances, such as cigarette smoke, cause cancer. Having this type of information could also be used to help people avoid coming into contact with cancer causing substances, since these substances would be proven, rather than merely suspected, of playing a role in oncogenesis.